ABSTRACT The inadequacy of current treatment options to cure infections caused by MDR Gram-negative pathogens results in high rates of morbidity and mortality along with their concomitant treatment costs. The goal of this proposal is to develop a series of pyranopyridine (PyPy) inhibitors of RND family efflux pumps, a major component of the MDR phenotype of Gram-negative pathogens, into an adjunctive therapy consisting of an optimized efflux pump inhibitor (EPI) and minocycline (MIN). The adjunctive therapy will be used to treat bloodstream infections caused by multidrug resistant (MDR) pathogens of the Enterobacteriaceae, including organisms designated as urgent and serious threats by the CDC, namely Carbapenem-Resistant and Extended Spectrum Beta Lactamase producing Enterobacteriaceae. PyPy EPIs are potent inhibitors of the major RND efflux pump (AcrB) in Escherichia coli and other Enterobacteriaceae, which extrude diverse classes of antibiotics from the periplasmic space to the exterior of the cell. Overexpression of RND efflux pumps plays an important factor in the MDR phenotype of a significant fraction of clinical isolates, which can be reversed by PyPy EPIs. Optimization of the PyPy series through SAR-driven and structure-based drug design has generated analogs that potentiate the antibacterial activity of several antibiotics which are substrates of AcrB at submicromolar concentrations in vitro. Our current lead, MBX-4191, is soluble in aqueous solutions (? 100 M), exhibits limited cytotoxicity (CC50 47 M), and a favorable in vitro ADME profile. MBX-4191 achieves high levels of exposure in mice (AUC = 26,000 hr*ng/mL at 10 mg/kg IV) and is well-tolerated after a single intravenous dose (MTD ?200 mg/kg), and after multiple IV doses (50 mg/kg, bid, 4d). MBX-4191 rescues the activity of MIN at 4 g/ml, the breakpoint concentration, against MIN-resistant strains of E. coli and K. pneumoniae in in vitro assays. Significantly, MBX- 4191 rescues the activity of MIN (50 mg/kg, BID) against a MIN-resistant, KPC+ strain of Klebsiella pneumoniae (MIN MIC = 32 g/ml) in a murine sepsis model of infection. In this project, we will chemically optimize the pyranopyridine EPI lead series in an SAR and structure-driven rational drug discovery effort focused on improving drug-like properties, pharmacokinetics, and efficacy in mice, while maintaining potency during years 1 and 2. In year 2, we will select a preclinical candidate and a back-up candidate for initial IND-enabling (GLP) pharmacokinetic, toxicology and safety pharmacology studies in rats. To enable preclinical studies, we will initiate manufacturing of a non-GMP lot (1 kg) of drug product. To achieve the goal of the proposed project, we will complete the following specific aims: AIM 1. Optimize lead series through SAR-driven analog generation. (years 1-2). AIM 2: Prioritize lead series analogs through in vitro biology and ADME evaluations (years 1-2). AIM 3: Select a preclinical candidate and backup based on in vivo properties (years 1-2). AIM 4: Conduct IND-enabling pharmacokinetic, toxicology and safety pharmacology studies (year 3). AIM 5: Chemistry, Manufacturing and Controls. (years 2-3).